Analysis of the principle of action of injection grade HEPES buffer system as mRNA stock buffer

In the 2016 BioNTech article on LPX mRNA formulations, HEPES was used as the buffer in the mRNA stock solution. In this study, HEPES buffer was used to prepare 1mg/ml RNA stock solution for further preparation of RNA-LPX.

HEPES has advantages as an IVT reaction system buffer, which is why we infer BioNTech uses HEPES buffer as the mRNA stock buffer based on relevant literature.

3、 The prominent features of HEPES compared to other buffering systems

1. The pKa changes very little with temperature, and the pH value of the buffer drifts very little with temperature.

2. HEPES has stable properties and does not interact with other ions. On the one hand, HEPES does not complex with other ions and affect their functions. On the other hand, the presence of other ions does not affect the buffering capacity of HEPES.

3. The maintenance of pH in cell culture medium mainly relies on the bicarbonate and 5% CO2 in the medium. When cells are in an open environment, CO2 escape can cause a rapid increase in pH beyond the physiological pH range, while adding HEPES buffer to the culture medium does not have this problem.

4. HEPES does not easily penetrate biological membranes and does not interfere with biochemical reactions within cells.

4、 Precautions when using HEPES buffer system

1. Due to the presence of paizine groups, when HEPES is exposed to light, it produces H2O2, which is toxic to cultured cells or other biologically active objects. At the same time, substances sensitive to redox reactions should not be used together. Therefore, the operation of HEPES as a buffer should be carried out under dark conditions as much as possible.

2. HEPES is commonly used in the biochemical field at concentrations of 10-25mM. When the concentration is below 10mM, the buffering capacity is weak and cannot achieve good buffering effect. When the concentration is high, it may have toxic effects on some cells.

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